For more than two decades, tremendous advances in regenerative medicine have provided hope that materials will offer solutions to organ and tissue shortages that occur worldwide. However, overcoming the remaining challenges will require many additional innovations, especially in materials that possess specific functionality that guide the regenerative processes.
Nanofibrous scaffolds possessing mechanical properties, porous microstructure, and dimensional similarity to collagen fibers have been used to mimic the natural extracellular matrix (ECM) and are highly relevant for tissue engineering in a number of different applications. Polymeric nanofibers have been fabricated into a variety of constructs and scaffolds using melt- or electrospinning processes that are able to control size, morphology and alignment by varying conditions including solvent, concentration, additives and electrode design.
For regenerative medicine applications, the polymeric precursors used to fabricate the nanofiber-based scaffolds should be both biocompatible and biodegradable. Many biodegradable and biocompatible polymers such as polyglycolic acid (PGA), poly(lactic acid) (PLA), poly(lactide-co-glycotide) (PLGA) and poly(ε-caprolactone) (PCL) have been widely investigated as fiber and nanofiber precursor materials. Although these degradable polymers meet several of the basic requirements for tissue engineering applications, bioactive molecules to guide cellular behavior and preserve cell phenotype are required for optimal performance. Specific functionalities that could guide or direct specific biological function need to be incorporated efficiently. There are generally two methods available for biomolecule functionalization: physical adsorption and chemical bonding. While physical adsorption risks the loss of biomolecules over time, chemical conjugation usually requires multi-step processing, and purification both of which often included harsh conditions.
Additionally, the derivitization of nanofibers often requires multiple procedures, including plasma treatment, wet chemical methods, surface graft polymerization, and co-electrospinning of surface active agents with polymers. Each of these modifications is time and resource intensive to optimize and may lead to immune specific reactions and biocompatibility problems.
A new method that enables efficient, orthogonal and bio-system friendly functionalization is preferred. Copper-catalysed click chemistry has been used for efficient functionalization of polymers. However, the side effect of copper ions leads to biocompatibility problems. Recently, the discovery of strain-promoted azide alkyne cycloaddition has provided a robust chemical method for the efficient conjugation of biomolecules. This method has been widely used in bioimaging and bioconjugation. The present invention makes beneficial use of this click chemistry and provides guidance for the creation of fibrous scaffolds and their post fabrication biofunctionalization through such azide alkyne cycloaddition chemistry.
Peptides, growth factors and carbohydrates have each been covalently tethered to the surfaces of synthetic and naturally-derived polymers to stimulate specific cell functions. Concerns about the bioavailability, specificity and activity of the tethered species persist. Recent studies show that the desired biological response can be obtained with the appropriate tether. A number of synthetic degradable polymers, including poly(lactic acid) are presently utilized clinically; but, cellular systems do not readily interact directly with synthetic polymers through normal integrin mediated assemblies. Tyr-Ile-Gly-Ser-Arg (YIGSR), a bio-active peptide derived from laminin, was shown to promote cell attachment and laminin receptor binding. Graf, J.; Ogle, R. C.; Robey, F. A.; Sasaki, M.; Martin, G. R.; Yamada, Y.; Kleinman, H. K. Biochemistry 1987, 26, 6896-6900. YIGSR-functionalized matrices have showed similar or superior cellular effects to Ile-Lys-Val-Ala-Val (IKVAV) peptide, a more commonly studied laminin-derived peptide and laminin-coated matrices. The art could benefit from incorporating YIGSR into nanofibers matrices to promote the directed differentiation of embryonic stem cells into neurons. However, the precise, regiospecific functionalization of degradable polymers with biological motifs capable of directing cellular function has been so complicated with regard to solvents, catalysts, residuals and processing methods, that they have been deemed translationally irrelevant. The recent evolution of click chemistry as a method to functionalize polymers and materials has enabled the derivatization of both natural and synthetic polymers in ways that were not previously possible.